This project focuses on the regulation and function of proteins of the secretory vesicle responsible for chemical coding of neurotransmission in mammalian neurons, including vesicular transporters for amine neurotransmitters, and co-stored secretory proteins and neuropeptides. Both isoforms of the human vesicular monoamine transporter (hVMAT1 and hVMAT2) are now cloned and characterized. HVMAT1 is expressed only in neuroendocrine cells, while only HVMAT2 is expressed in neurons. HVMAT2 is also expressed in histaminocytes of human stomach, consistent with histamine's higher affinity for hVMAT2 than hVMAT1. Neuropharmacological and neurotoxic agents like amphetamine, fenfluramine, and MPP+ exhibit a high affinity for hVMAT2, suggesting that they interact with VMAT2 as well as plasma membrane amine carriers in vivo. An efficient cell-free vesicular acetylcholine transport assay has been developed to study the structural basis for recognition of acetylcholine by the vesicular acetylcholine transporter, VAChT. Antibodies directed to human VAChT visualize cholinergic nerve terminals in routinely fixed human tissues, providing new information about the chemical neuroanatomy of the human cholinergic nervous system, including a subatnatial cholinergic innervation of the median eminence of the hypothalamus. VAChT and VMAT2 are differentially targeted in synaptic vesicle-containing cell lines engineered to express both transporters. This suggests that cholinergic and monoaminergic synaptic vesicles are biosynthetically distinct organelles, an additional dimension in specificity of chemical coding in monoaminergic versus cholinergic neurons. A 400 bp enhancer (TSE: tissue-specific enhancer) has been defined in the human gene encoding VIP, a neuropeptide co-stored with acetylcholine. It contains repeated motifs related to OCT and LIM protein binding sequences, and requires the presence of a proximal cyclic AMP-response element in the VIP gene for full expression. In addition to their cell-specific coding to individual neurons, neuropeptides are up-regulated in response to increased secretory activity (stimulus-secretion-synthesis coupling). In chromaffin cells expressing and secreting enkephalin, this process has now been shown to occur via a non-immediate early gene (IEG)-mediated mechanism distinguishing it from neuropeptide gene regulation mediated by protein kinase A and C, which require new protein synthesis.